Subjects

Nup98 is a glycine-leucine-phenylalanine-glycine (GLFG) repeat-containing nucleoporin that, in addition to nuclear transport, contributes to multiple aspects of gene regulation. Previous studies revealed its dynamic localization within intranuclear structures known as GLFG bodies. Here we show that the mammalian Nup107-160 complex (Y-complex), a major scaffold module of the nuclear pore, together with its partner Elys, colocalizes with Nup98 in GLFG bodies. The frequency and size of GLFG bodies vary among HeLa sublines, and we find that an increased level of Nup98 is associated with the presence of bodies. Recruitment of the Y-complex and Elys into GLFG bodies requires the C-terminal domain of Nup98. During cell division, Y-Nup-containing GLFG bodies are disassembled in mitotic prophase, significantly ahead of nuclear pore disassembly. FRAP studies revealed that, unlike at nuclear pores, the Y-complex shuttles into and out of GLFG bodies. Finally, we show that within the nucleoplasm, a fraction of Nup107, a key component of the Y-complex, displays reduced mobility, suggesting interaction with other nuclear components. Together our data uncover a previously neglected intranuclear pool of the Y-complex that may underscore a yet-uncharacterized function of these nucleoporins inside the nucleus, even in cells that contain no detectable GLFG bodies.

Subjects

The localization of β-actin mRNA to the leading lamellae of chicken fibroblasts and neurite growth cones of developing neurons requires a 54-nt localization signal (the zipcode) within the 3′ untranslated region. In this study we have identified and isolated five proteins binding to the zipcode. One of these we previously identified as zipcode binding protein (ZBP)1, a 4-KH domain protein. A second is now investigated in detail: a 92-kD protein, ZBP2, that is especially abundant in extracts from embryonic brain. We show that ZBP2 is a homologue of the human hnRNP protein, KSRP, that appears to mediate premRNA splicing. However, ZBP2 has a 47-amino acid (aa) sequence not present in KSRP. Various portions of ZBP2 fused to GFP indicate that the protein most likely shuttles between the nucleus and the cytoplasm, and that the 47-aa insert promotes the nuclear localization. Expression of a truncated ZBP2 inhibits the localization of β-actin mRNA in both fibroblast and neurons. These data suggest that ZBP2, although predominantly a nuclear protein, has a role in the cytoplasmic localization of β-actin mRNA.

Subjects

We used structured illumination microscopy (SIM) to obtain super-resolution images of muscle attachment structures in Caenorhabditis elegans striated muscle. SIM imaging of M-line components revealed two patterns: PAT-3 (β-integrin) and proteins that interact in a complex with the cytoplasmic tail of β-integrin and localize to the basal muscle cell membrane [UNC-112 (kindlin), PAT-4 (ILK), UNC-97 (PINCH), PAT-6 (α-parvin), and UNC-95], are found in discrete, angled segments with gaps. In contrast, proteins localized throughout the depth of the M-line (UNC-89 (obscurin) and UNC-98) are imaged as continuous lines. Systematic immunostaining of muscle cell boundaries revealed that dense body components close to the basal muscle cell membrane also localize at cell boundaries. SIM imaging of muscle cell boundaries reveal “zipper-like” structures. Electron micrographs reveal electron dense material similar in appearance to dense bodies located adjacent to the basolateral cell membranes of adjacent muscle cells separated by ECM. Moreover, by EM, there are a variety of features of the muscle cell boundaries that help explain the zipper-like pattern of muscle protein localization observed by SIM. Short dense bodies in atn-1 mutants that are null for α-actinin and lack the deeper extensions of dense bodies, showed “zipper-like” structures by SIM similar to cell boundary structures, further indicating that the surface-proximal components of dense bodies form the “zipper-like” structures at cell boundaries. Moreover, mutants in thin and thick filament components do not have “dot-like” dense bodies, suggesting that myofilament tension is required for assembly or maintenance of proper dense body shape.

Subjects

The glucocorticoid receptor (GR) is a constitutively expressed transcriptional regulatory factor (TRF) that controls many distinct gene networks, each uniquely determined by particular cellular and physiological contexts. The precision of GR-mediated responses seems to depend on combinatorial, context-specific assembly of GR-nucleated transcription regulatory complexes at genomic response elements. In turn, evidence suggests that context-driven plasticity is conferred by the integration of multiple signals, each serving as an allosteric effector of GR conformation, a key determinant of regulatory complex composition and activity. This structural and mechanistic perspective on GR regulatory specificity is likely to extend to other eukaryotic TRFs.

Subjects

Protein phosphatase 2A (PP2A) is a heterotrimer composed of single catalytic and scaffolding subunits and one of several possible regulatory subunits. We identified PPTR-2, a regulatory subunit of PP2A, as a binding partner for the giant muscle protein UNC-89 (obscurin) in Caenorhabditis elegans. PPTR-2 is required for sarcomere organization when its paralogue, PPTR-1, is deficient. PPTR-2 localizes to the sarcomere at dense bodies and M-lines, colocalizing with UNC-89 at M-lines. PP2A components in C. elegans include one catalytic subunit LET-92, one scaffolding subunit (PAA-1), and five regulatory subunits (SUR-6, PPTR-1, PPTR-2, RSA-1, and CASH-1). In adult muscle, loss of function in any of these subunits results in sarcomere disorganization. rsa-1 mutants show an interesting phenotype: One of the two myosin heavy chains, MHC A, localizes as closely spaced double lines rather than single lines. This "double line" phenotype is found in rare missense mutants of the head domain of MHC B myosin, such as unc-54(s74). Analysis of phosphoproteins in the unc-54(s74) mutant revealed two additional phosphoserines in the nonhelical tailpiece of MHC A. Antibodies localize PPTR-1, PAA-1, and SUR-6 to I-bands and RSA-1 to M-lines and I-bands. Therefore, PP2A localizes to sarcomeres and functions in the assembly or maintenance of sarcomeres.

Subjects

UNC-89 is a giant polypeptide located at the sarcomeric M-line of Caenorhabditis elegans muscle. The human homologue is obscurin. To understand how UNC-89 is localized and functions, we have been identifying its binding partners. Screening a yeast two-hybrid library revealed that UNC-89 interacts with paramyosin. Paramyosin is an invertebrate-specific coiled-coil dimer protein that is homologous to the rod portion of myosin heavy chains and resides in thick filament cores. Minimally, this interaction requires UNC- 89's SH3 domain and residues 294-376 of paramyosin and has a KD of ∼1.1 μM. In unc-89 loss-of-function mutants that lack the SH3 domain, paramyosin is found in accumulations. When the SH3 domain is overexpressed, paramyosin is mislocalized. SH3 domains usually interact with a proline-rich consensus sequence, but the region of paramyosin that interacts with UNC-89's SH3 is α-helical and lacks prolines. Homology modeling of UNC-89's SH3 suggests structural features that might be responsible for this interaction. The SH3-binding region of paramyosin contains a "skip residue," which is likely to locally unwind the coiled-coil and perhaps contributes to the binding specificity.

Subjects

We have identified the gene C28H8.6 (pxl-1) as the Caenorhabditis elegans orthologue of vertebrate paxillin. PXL-1 contains the four C-terminal LIM domains conserved in paxillin across all species and three of the five LD motifs found in the N-terminal half of most paxillins. In body wall muscle, PXL-1 antibodies and a full-length green fluorescent protein translational fusion localize to adhesion sites in the sarcomere, the functional repeat unit in muscle responsible for contraction. PXL-1 also localizes to ring-shaped structures near the sarcolemma in pharyngeal muscle corresponding to podosome-like sites of actin attachment. Our analysis of a loss-of-function allele of pxl-1, ok1483, shows that loss of paxillin leads to early larval arrested animals with paralyzed pharyngeal muscles and eventual lethality, presumably due to an inability to feed. We rescued the mutant phenotype by expressing paxillin solely in the pharynx and found that these animals survived and are essentially wild type in movement and body wall muscle structure. This indicates a differential requirement for paxillin in these two types of muscle. In pharyngeal muscle it is essential for contraction, whereas in body wall muscle it is dispensable for filament assembly, sarcomere stability, and ultimately movement.

Subjects

In vertebrates, zyxin is a LIM-domain protein belonging to a family composed of seven members. We show that the nematode Caenorhabditis elegans has a unique zyxin-like protein, ZYX-1, which is the orthologue of the vertebrate zyxin subfamily composed of zyxin, migfilin, TRIP6, and LPP. The ZYX-1 protein is expressed in the striated body-wall muscles and localizes at dense bodies/Z-discs and M-lines, as well as in the nucleus. In yeast two-hybrid assays ZYX-1 interacts with several known dense body and M-line proteins, including DEB-1 (vinculin) and ATN-1 (α-actinin). ZYX-1 is mainly localized in the middle region of the dense body/Z-disk, overlapping the apical and basal regions containing, respectively, ATN-1 and DEB-1. The localization and dynamics of ZYX-1 at dense bodies depend on the presence of ATN-1. Fluorescence recovery after photobleaching experiments revealed a high mobility of the ZYX-1 protein within muscle cells, in particular at dense bodies and M-lines, indicating a peripheral and dynamic association of ZYX-1 at these muscle adhesion structures. A portion of the ZYX-1 protein shuttles from the cytoplasm into the nucleus, suggesting a role for ZYX-1 in signal transduction. We provide evidence that the zyx-1 gene encodes two different isoforms, ZYX-1a and ZYX-1b, which exhibit different roles in dystrophin-dependent muscle degeneration occurring in a C. elegans model of Duchenne muscular dystrophy.

Subjects

The thiazide-sensitive sodium chloride cotransporter (NCC) and the epithelial sodium channel (ENaC) are two of the most important determinants of salt balance and thus systemic blood pressure. Abnormalities in either result in profound changes in blood pressure. There is one segment of the nephron where these two sodium transporters are coexpressed, the second part of the distal convoluted tubule. This is a key part of the aldosterone-sensitive distal nephron, the final regulator of salt handling in the kidney. Aldosterone is the key hormonal regulator for both of these proteins. Despite these shared regulators and coexpression in a key nephron segment, associations between these proteins have not been investigated. After confirming apical localization of these proteins, we demonstrated the presence of functional transport proteins and native association by blue native PAGE. Extensive coimmunoprecipitation experiments demonstrated a consistent interaction of NCC with α-And γ-ENaC. Mammalian two-hybrid studies demonstrated direct binding of NCC to ENaC subunits. Fluorescence resonance energy transfer and immunogold EM studies confirmed that these transport proteins are within appropriate proximity for direct binding. Additionally, we demonstrate that there are functional consequences of this interaction, with inhibition of NCC affecting the function of ENaC. This novel finding of an association between ENaC and NCC could alter our understanding of salt transport in the distal tubule.